Sedentary and microbial organisms in all environments—marine, terrestrial, and fresh water—must produce secondary metabolites with which they can interact with the world around them. Micro-environments, such as fresh water ponds, inner-city forests, or coastal estuaries, to name a few, may be home to countless organisms that must respond to incredibly localized stressors that make no two environments exactly the same. New chemical structures are emerging all the time from countless environmental sources, and as threats to human health evolve, it could certainly be argued that natural products research is the way forwards.
Thanks to genomic advancements, it is clear that micro-organisms cultured in the lab routinely produce only a fraction of the secondary metabolites that are coded for in their DNA. (Gross, H. Strategies to Unravel the Function of Orphan Biosynthesis Pathways: Recent Examples and Future Prospects. Applied Microbiology and Biotechnology. 2007, pp 267-277). This is accomplished by the regulation of transcription by enzymes activated and deactivated based on environmental factors. (Bok, J. W.; Keller, N. P. LaeA, a Regulator of Secondary Metabolism in Aspergillus Spp. Eukaryot. Cell 2004, 3 (2), 527-535). In filamentous fungi, it is known that most secondary metabolite genes are clustered to allow for the most efficient regulation and these clusters can be activated or deactivated by culture conditions, resulting in vastly different metabolite production. (Bok, J. W.; Keller, N. P. LaeA, a Regulator of Secondary Metabolism in Aspergillus Spp. Eukaryot. Cell 2004, 3 (2), 527-535; Keller, N. P.; Hohn, T. M. REVIEW Metabolic Pathway Gene Clusters in Filamentous Fungi. Fungal Genet. Biol. 1997, 21, 17-29).
Once isolated, a micro-organism of interest can be cultured in the lab under any number of easily accessible stressors that can change secondary metabolite production. Culture variations can be as simple as changing the shape of the culture vessel, or as complex as the addition of biological material from another microbe or host organism. In this way, a single strain can produce a multitude of different compounds. (Gross, H. Strategies to Unravel the Function of Orphan Biosynthesis Pathways: Recent Examples and Future Prospects. Applied Microbiology and Biotechnology. 2007, pp 267-277; Lim, F. Y.; Sanchez, J. F.; Wang, C. C. C.; Keller, N. P. Toward Awakening Cryptic Secondary Metabolite Gene Clusters in Filamentous Fungi. Methods Enzymol. 2012, 517, 303-324; Brakhage, A. A.; Schroeckh, V. Fungal Secondary Metabolites—Strategies to Activate Silent Gene Clusters. Fungal Genet. Biol. 2011, 48 (1), 15-22; Bode, H. B.; Bethe, B.; Hofs, R.; Zeeck, A. Big Effects from Small Changes: Possible Ways to Explore Nature's Chemical Diversity. ChemBioChem 2002, 3 (7), 619-627; Scherlach, K.; Hertweck, C. Triggering Cryptic Natural Product Biosynthesis in Microorganisms. Org. Biomol. Chem. 2009, 7 (9), 1753-1760). While this ‘OSMAC’ (‘One Strain Many Compounds’) strategy is extremely useful in exploiting the full biosynthetic potential of a micro-organism of interest, it is rather intensive in time and consumables. (Bode, 2002).
Rather than systematically changing culture conditions, the biosynthetic potential of a micro-organism of interest can also be explored through whole genome sequencing. Many secondary metabolites are products of known biosynthetic pathways. The ability to ascribe a product to the genes that code for it allows for the unique ability to analyze a whole genome and predict the metabolites that can be produced. Culture conditions curated to that biosynthetic pathway can then be employed to isolate specific compounds of interest. (Bromann, K.; Toivari, M.; Viljanen, K.; Vuoristo, A.; Ruohonen, L.; Nakari-Setälä, T. Identification and Characterization of a Novel Diterpene Gene Cluster in Aspergillus nidulans. PLoS One 2012, 7 (4); Bergmann, S.; Schümann, J.; Scherlach, K.; Lange, C.; Brakhage, A. A.; Hertweck, C. Genomics—Driven Discovery of PKS—NRPS Hybrid Metabolites from Aspergillus nidulans. Nat. Chem. Biol. 2007, 3 (4), 213-217; Scherlach, K.; Hertweck, C. Discovery of Aspoquinolones A-D, Prenylated Quinoline-2-One Alkaloids from Aspergillus nidulans, Motivated by Genome Mining. Org. Biomol. Chem. 2006, 4, 3517-3520; Van Lanen, S. G.; Shen, B. Microbial Genomics for the Improvement of Natural Product Discovery. Curr. Opin. Microbiol. 2006, 9 (3), 252-260; Cone, C.; Challis, G. L. New Natural Product Biosynthetic Chemistry Discovered by Genome Mining. Nat. Prod. Rep. 2009, 26 (8), 977-986; Challis, G. L. Mining Microbial Genomes for New Natural Products and Biosynthetic Pathways. Microbiology 2008, 154 (6), 1555-1569).
Genome mining and the OSMAC approach are both useful techniques for the discovery of the biosynthetic potential of a single organism. If, however, you have a microbial library that you would like to screen, these techniques may not be the most efficient. Epigenetic modification—that is, the use of small molecule enzyme inhibitors to promote the expression and prevent the silencing or downregulation of secondary metabolite gene clusters can be used as a more ubiquitous technique to exploit the biosynthetic potential of a larger number of microorganisms. (Williams, R. B.; Henrikson, J. C.; Hoover, A. R.; Lee, A. E.; Cichewicz, R. H. Epigenetic Remodeling of the Fungal Secondary Metabolome. Org Biomol Chem 2008, 6 (11), 1895-1897; Cichewicz, R. H. Epigenome Manipulation as a Pathway to New Natural Product Scaffolds and Their Congeners Robert. Nat. Prod. Rep. 2010, 27 (1), 11-22; Henrikson, J. C.; Hoover, A. R.; Joyner, P. M.; Cichewicz, R. H. A Chemical Epigenetics Approach for Engineering the in Situ Biosynthesis of a Cryptic Natural Product from Aspergillus niger. Org. Biomol. Chem. 2009, 7 (3), 435-438; Wang, X.; Sena Filho, J. G.; Hoover, A. R.; King, J. B.; Ellis, T. K.; Powell, D. R.; Cichewicz, R. H. Chemical Epigenetics Alters the Secondary Metabolite Composition of Guttate Excreted by an Atlantic-Forest-Soil-Derived Penicillium citreonigrum. J. Nat. Prod. 2010, 73 (5), 942-948). Histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors can be used as culture additives to epigenetically ‘turn on’ secondary metabolite gene clusters in a library of filamentous fungi for the maximum surveying of bioactive natural product potential therein. (Beau, J.; Mahid, N.; Burda, W. N.; Harrington, L.; Shaw, L. N.; Mutka, T.; Kyle, D. E.; Barisic, B.; Van Olphen, A.; Baker, B. J. Epigenetic Tailoring for the Production of Anti-Infective Cytosporones from the Marine Fungus Leucostoma persoonii. Mar. Drugs 2012, 10 (4), 762-774).
There are many different techniques available for natural products drug discovery efforts. While exploring the biosynthetic potential of a single organism can be very lucrative, screening efforts are needed in order to identify those “lead” organisms. With a robustly designed screening program, natural product extracts from a multitude of sources can be screened side by side in high-throughput capable bioassays against a wide variety of disease targets. The resulting diversity of bioactivity information combined with metabolite profiling can afford intense prioritization of extracts at a very early stage, streamlining further chemical investigation to a highly time and cost effective level of efficiency.
Drug Discovery Targets
Natural products isolation efforts largely follow the same generic scheme (FIG. 1). Efforts aimed at drug discovery can take place at any of the stages, from extraction to pure compound isolation. There are pros and cons to each approach, though it is generally accepted that the earlier the efforts can be prioritized, the better.
Crude extracts can contain thousands of compounds, however, it is possible to get useful information from that complex mixture in a high-throughput way. Metabolite profiling of crude extracts can be used for initial dereplication and more advanced matabolomic analysis can reveal chemical outliers that may be of interest. (Sica, V. P.; Raja, H. A.; El-Elimat, T.; Kertesz, V.; Van Berkel, G. J.; Pearce, C. J.; Oberlies, N. H. Dereplicating and Spatial Mapping of Secondary Metabolites from Fungal Cultures in Situ. J. Nat. Prod. 2015, 78 (8), 1926-1936; Kellogg, J. J.; Todd, D. A.; Egan, J. M.; Raja, H. A.; Oberlies, N. H.; Kvalheim, O. M.; Cech, N. B. Biochemometrics for Natural Products Research: Comparison of Data Analysis Approaches and Application to Identification of Bioactive Compounds. J. Nat Prod. 2016, 79 (2), 376-386). High-throughput bioassays that are tolerant of complex mixtures can be used to discover and prioritize activity early in the investigation process. More sensitive and selective bioassays that are not tolerant of complex mixtures would require more purified fractions or pure compounds. It is important, therefore, when embarking on a natural products screening program, to coordinate bioassay capabilities to isolation protocols, in addition to other target selection criteria. The targets described below are of great contemporary relevance to human health concerns and each feature robust bioassay methodologies that assist in early crude extract level prioritization.
The ESKAPE Pathogens
With growing antibiotic resistance, and a decrease in antibiotic drug discovery, the Infectious Disease Society of America issued a ‘call to arms’ in 2009 to the drug discovery community to combat what they called the ESKAPE pathogens: the gram positive Enterococcus faecium and Staphylococcus aureus, and gram negative Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. (Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious Diseases Society of America. Clin. Infect. Dis. 2009, 48 (1), 1-12). These clinically relevant, highly drug resistant pathogens represent a continuously growing threat to human health and an important target for drug discovery efforts. (Pogue, J. M.; Kaye, K. S.; Cohen, D. A.; Marchaim, D. Appropriate Antimicrobial Therapy in the Era of Multidrug-Resistant Human Pathogens. Clin. Microbiol. Infect. 2015, 21 (4), 302-312; Fleeman, R.; Lavoi, T. M.; Santos, R. G.; Morales, A.; Nefzi, A.; Welmaker, G. S.; Medina-Franco, J. L.; Giulianotti, M. A.; Houghten, R. A.; Shaw, L. N. Combinatorial Libraries as a Tool for the Discovery of Novel, Broad-Spectrum Antibacterial Agents Targeting the ESKAPE Pathogens. J. Med. Chem. 2015, 58 (8), 3340-3355).
Leishmania donovani 
Cutaneous Leishmaniasis accounts for one million cases annually with 310 million people being at risk for contraction. Visceral Leishmaniasis accounts for 300,000 cases annually which result in 20,000 deaths annually. A neglected tropical disease (NTD), Leishmaniasis is a parasitic infection caused by an intramacrophage protozoa that is transmitted to humans by the bite of infected sandflies. The visceral form of this disease, most commonly caused by Leishmania donovani, is typically fatal when left untreated. Upon entering the host, the parasite—in a non-flagellated amastigote life stage—invades macrophage cells to travel through the body and reproduce. (Pulvertaft, R.; Hoyle, G. Stages in the Life-Cycyle of Leishmania donovani. Trans R Soc Trop Med Hyg 1960, 54, 191-196). Recent advances in infected macrophage in-vitro culture techniques allow for more clinically relevant assays to be performed in a high throughput screening (HTS) context. (Annang, F.; Perez-Moreno, G.; Garcia-Hernandez, R.; Cordon-Obras, C.; Martin, J.; Tormo, J. R.; Rodriguez, L.; de Pedro, N.; Gomez-Perez, V.; Valente, M.; Reyes, F.; Genilloud, O.; Vicente, F.; Castanys, S.; Ruiz-Perez, L. M.; Navarro, M.; Gamarro, F.; Gonzalez-Pacanowska, D. High-Throughput Screening Platform for Natural Product-Based Drug Discovery against 3 Neglected Tropical Diseases: Human African Trypanosomiasis, Leishmaniasis, and Chagas Disease. J. Biomol. Screen. 2015, 20 (1), 82-91; De Rycker, M.; Hallyburton, I.; Thomas, J.; Campbell, L.; Wyllie, S.; Joshi, D.; Cameron, S.; Gilbert, I. H.; Wyatt, P. G.; Frearson, J. A.; Fairlamb, A. H.; Gray, D. W. Comparison of a High-Throughput High-Content Intracellular Leishmania donovani Assay with an Axenic Amastigote Assay. Antimicrob. Agents Chemother. 2013, 57 (7), 2913-2922; Siqueira-Neto, J. L.; Moon, S.; Jang, J.; Yang, G.; Lee, C.; Moon, H. K.; Chatelain, E.; Genovesio, A.; Cechetto, J.; Freitas-Junior, L. H. An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages. PLoS Negl. Trop. Dis. 2012, 6 (6)). These advancements will hopefully aid in the discovery of new treatments for this disease in the face of increasing resistance to existing treatments. (Balasegaram, M.; Ritmeijer, K.; Lima, M. A.; Burza, S.; Ortiz Genovese, G.; Milani, B.; Gaspani, S.; Potet, J.; Chappuis, F. Liposomal Amphotericin B as a Treatment for Human Leishmaniasis. Expert Opin. Emerg. Drugs 2012, 17 (4), 493-510).
Mycobacterium tuberculosis 
Tuberculosis (TB) remains a global health crisis, despite the advances of the whole genome sequencing project that revealed the genome of Mycobacterium tuberculosis. This disease, whose latent form is estimated to infect one third of the world's population, poses many drug development hurdles. Multi-drug resistant (MDR-TB) and extensively drug resistant (XDRTB) strains have emerged despite the current course of treatment typically involving combinatorial therapies aimed directly at preventing resistance. Drug discovery efforts, therefore, must address new mechanisms of action or M. tuberculosis targets. Additionally, TB drugs have the burden of needing to be compatible in combinatorial treatments for the immunocompromised, particularly those with HIV/AIDS, among whom incidence of this disease are highest. (Lechartier, B.; Rybniker, J.; Zumla, A.; Cole, S. T. Tuberculosis Drug Discovery in the Post-Post-Genomic Era. EMBO Mol. Med. 2014, 6 (2), 1-11). Natural products based drug discovery against this target have revealed promising results, with many existing treatments coming from natural products. With such a demanding target comes the need to screen a broad swath of chemical space, confirming natural products drug discovery efforts as a promising way forward in the search for treatments of this disease. (Mdluli, K.; Kaneko, T.; Upton, A. The Tuberculosis Drug Discovery and Development Pipeline and Emerging Drug Targets. Cold Spring Harb Perspect Med 2015, 5).
Clostridium difficile 
The leading cause of healthcare related infection, Clostridium difficile is an easily spread, diarrhea causing bacteria that is considered a threat to human health worldwide. The use of antibiotics which upset the human gut microbiome is the primary cause of C. difficile infection (CDI), but any immunocompromised individuals are at risk. With increasing incidences of resistance, recurrence, and mortality, the need for discovery of new treatments against this bacteria is imperative. Most challengingly, new drugs to fight CDI must act without impact on the normal human gut fauna. Many novel treatment avenues have been suggested, among which, the discovery and use of bacterial natural products remain of high interest. (Zucca, M.; Scutera, S.; Savoia, D. Novel Avenues for Clostridium difficile Infection Drug Discovery. Expert Opin. Drug Discov. 2013, 8 (4), 459-477; Suwantarat, N.; Bobak, D. A. Current Status of Nonantibiotic and Adjunct Therapies for Clostridium difficile Infection. Curr. Infect. Dis. Rep. 2011, 13 (1), 21-27; Spigaglia, P. Recent Advances in the Understanding of Antibiotic Resistance in Clostridium difficile Infection. Ther. Adv. Infect. Dis. 2016, 3 (1), 23-42).
Naegleria fowleri 
Naegleria fowleri is a free living, warm-water loving amoeba that causes the nearly always fatal primary amoebic meningoencephalitis (PAM). Diagnosis of PAM is extremely difficult, and current treatment options are time sensitive and limited to existing drug combinations (e.g. Amphotericin B, fluconazole, and miltefosine). (Capewell, L. G.; Harris, A. M.; Yoder, J. S.; Cope, J. R.; Eddy, B. A.; Roy, S. L.; Visvesvara, G. S.; Fox, L. M.; Beach, M. J. Diagnosis, Clinical Course, and Treatment of Primary Amoebic Meningoencephalitis in the United States, 1937-2013; YODER, J. S.; EDDY, B. A.; VISVESVARA, G. S.; CAPEWELL, L.; BEACH, M. J. The Epidemiology of Primary Amoebic Meningoencephalitis in the USA, 1962-2008. Epidemiol. Infect. 2010, 138 (7), 968-975). As clinicians start to understand and diagnose PAM better, and the risk of this disease continues to rise with increasing global temperatures, new drugs that specifically target this amoeba are urgently needed.
Cancer Targets
With structures as diverse as their targets, natural products have long played a role in the treatment of various cancers. (Newman, D. J.; Cragg, G. M. Natural Products as Sources of New Drugs from 1981 to 2014. J. Nat. Prod. 2016, 79 (3), 629-661; Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. Plant Antitumor Agents. VI. Isolation and Structure of Taxol, a Novel Antileukemic and Antitumor Agent from Taxus brevifolia. J. Am. Chem. Soc. 1971, 93 (9), 2325-2327; Fenical, W.; Jensen, P.; Kauffman, C.; Mayhead, S.; Faulkner, D.; Sincich, C.; Rao, M.; Kantorowski, E.; West, L.; Strangman, W.; Shimizu, Y.; Li, B.; Thammana, S.; Drainville, K.; Davies-Coleman, M.; Kramer, R.; Fairchild, C.; Rose, W.; Wild, R.; Vite, G.; Peterson, R. New Anticancer Drugs from Cultured and Collected Marine Organisms. Pharm. Biol. 2003, 41 (sup1), 6-14). As understanding of the complex physiology of human cells (both healthy and cancerous) continues to grow, assays directed at testing compounds against highly specific cellular targets continue to emerge. Rather than whole cancer-cell assays, these target specific assays can help to exclude compounds that are broadly cytotoxic to all cells in favor of compounds that are active within the specific mechanism of action that is desired. Autopalmitoylation dysregulation is implicated in many disease states. In a newly developed assay, palmitoylation of proteins can be monitored for modulation by compounds of interest in a HTS manner. This allows compounds to be rapidly screened not for their effect on the whole cell, but rather just on this particular pathway of interest. (Hamel, L. D.; Deschenes, R. J.; Mitchell, D. A. A Fluorescence-Based Assay to Monitor Autopalmitoylation of zDHHC Proteins Applicable to High-Throughput Screening Q. Anal. Biochem. 2014, 460, 1-8).